The organization of cells into subcellular compartments that are highly specialized for specific metabolic functions is a basic property of all eukaryotic cells. This compartmentalization relies on accurate targeting and translocation of proteins into specific organelles. The molecular basis of protein targeting to the hydrogenosome, an organelle found in the human-infective parasite Trichomonas vaginalis, is the focus of this proposal. This organelle plays a central role in the carbohydrate metabolism and drug susceptibility of T. vaginalis - a common etiologic agent of vaginitis. Hydrogenosomes provide a target for the selective killing of this parasite, as the organelle is not found in the host. Thus, these studies may provide a framework for future medical applications. We have developed an in vitro assay using purified T. vaginalis hydrogenosomes and radiolabelled proteins to study the translocation of proteins into this organelle. Results obtained during the first grant period indicate that (1) hydrogenosomal proteins are synthesized on free ribosomes and posttranslationally translocated into the organelle (2) translocation is dependent upon the presence of an amino terminal leader sequence (3) this leader sequence is cleaved upon translocation of proteins into hydrogenosomes, (4) translocation requires ATP and cytosolic factors (5) the hydrogenosomal protein, ferredoxin, associates with yeast mitochondria in vivo and (6) the amino terminal leader on hydrogenosomal proteins is similar to presequences that direct proteins into mitochondria. In this proposal we focus on three aspects of protein targeting to hydrogenosomes: (1) the essential biochemical properties of the leader sequence that allows it to function as a targeting signal (2) cytosolic and hydrogenosomal proteins which play a role in translocation and (3) identification of hydrogenosomal membrane proteins and characterization of their role in translocation. To this end, we will (a) examine the effect of mutating conserved residues of the leader sequence on protein translocation using our in vitro assay (b) determine whether protein translocation into hydrogenosomes requires a membrane potential (c) determine if cytosol from mitochondria and nonmitochondria containing cells can substitute for T. vaginalis cytosol (d) analyze putative chaperonins and other cytosolic proteins involved in translocation and (e) characterize hydrogenosomal membrane proteins. This detailed analysis of hydrogenosomal leader sequences and factors which play a role in protein translocation will provide insights into the origin and biogenesis of this organelle.